Cayae1 polynucleotides

ABSTRACT

The invention provides caYAE1 polypeptides and DNA (RNA) encoding such caYAE1 and a procedure for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing such caYAE1 for the treatment of infection, particularly fungal infections. Antagonists against such caYAE1 and their use as a therapeutic to treat infections, particularly fungal infections are also provided. Further provided are diagnostic assays for detecting diseases related to the presence of caYAE1 nucleic acid sequences and the polypeptides in a host. Also provided are diagnostic assays for detecting polynucleotides encoding caYAE1 and for detecting the polypeptide in a host.

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of these polynucleotides andpolypeptides; processes for making these polynucleotides and thesepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of these polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides derived from a Candida albicans gene,hereinafter all such polynucleotides and polypeptides are referred to as"caYAE1".

BACKGROUND OF THE INVENTION

The Saccharomyces cerevisiae genome project has identified many unknown,hypothetical proteins (EMBO Journal 15, 2031-2049, 1996). Thesesequences may be a useful starting point for the discovery ofbiologically important sequences. Using these sequences as researchtools to discover essential genes is one of their uses. Discovering andunderstanding essential genes in pathogenic organisms provides reagentsto screen for antipathogen compounds.

Many techniques are available to identify genes which are essential tosurvival per se, or essential to the establishment/maintenance of aninfection. These techniques include, for example, differential display(Chuang et al., J. Bacteriol. 175:2026-2036 (1993)), generation ofconditional lethal mutants by transposon mutagenesis (de Lorenzo, V. etal., Gene 123:17-24 (1993); Neuwald, A. F. et al., Gene 125:69-73(1993); and Takiff, H. E. et al., J. Bacteriol.174:1544-1553(1992)), and generation of conditional lethal mutants bychemical mutagenesis (Beckwith, J., Methods in Enzymology 204:3-18(1991)). Certain of these techniques were applied to discover thegenetic sequences of the present invention, caYAE1. Clearly, there is aneed for factors, such as essential polynucleotide and polypeptides fromfungal pathogens, since they may be used to screen compounds forantifungal activity and which may also be used to determine their rolesin pathogenesis of infection, dysfunction and disease. There is a need,therefore, for identification and characterization of such factors whichcan play a role in preventing, ameliorating or correcting infections,dysfunctions or diseases.

The polypeptide of the present invention has amino acid sequencehomology to known essential protein YAE1 of Saccharomyces cerevisiaehereinafter referred to as "scYAE1."

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelcaYAE1 peptides by homology between the amino acid sequence set out inFIG. 2 and known amino acid sequences of other proteins such as scYAE1protein.

It is a further object of the invention, moreover, to providepolynucleotides that encode caYAE1 polypeptides, particularlypolynucleotides that encode the polypeptide herein designated caYAE1.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding caYAE1 polypeptides inthe sequence set out in FIG. 1 SEQ ID NO:1!, or a fragment, analogue orderivative thereof.

In another particularly preferred embodiment of the present inventionthere is a novel protein from Candida albicans comprising the amino acidsequence of FIG. 2 SEQ ID NO:2!, or a fragment, analogue or derivativethereof.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding Candida albicans YAE1,including mRNAs, cDNAs, genomic DNAs and, in further embodiments of thisaspect of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful variants, analogsor derivatives thereof, or fragments thereof, including fragments of thevariants, analogs and derivatives, and compositions comprising same.

In accordance with another aspect of the present invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of caYAE1 andpolypeptides encoded thereby.

In accordance with this aspect of the invention there are provided novelpolypeptides of Candida referred to herein as caYAE1 as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful fragments, variants and derivatives thereof,variants and derivatives of the fragments, and analogs of the foregoing,and compositions comprising same.

Among the particularly preferred embodiments of this aspect of theinvention are variants of caYAE1 polypeptide encoded by naturallyoccurring alleles of the caYAE1 gene.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned caYAE1 polypeptides.

In accordance with yet another aspect of the present invention, thereare provided inhibitors to such polypeptides, useful as antifungalagents, including, for example, antibodies.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia: assessing caYAE1 expression; to treat, for example, candidosis(candidiasis), for example, superficial candidosis; candidosis of theoropharynx, such as, oral thrush (acute pseudomembraneous candodisis),denture stomatitis, angular choilitis (perleche), Candida leukoplakia(chronic hyperplastic candidosis), midline glossitis (median rbomboidglossitis, glossal central papillary atrophy, "antibiotic sore tongue",acute atrophic candidosis), and miscellaneous forms of oral candidosis;candidosis of the genitalia, such as, vulvovaginal candidosis (vaginalthrush, Candida colpitis), and candidosis of the penis (Candidabalanitis, balanoposthitis and urethritis); candidosis of the skin,nails and other external sites, such as, Candida intertrigo, napkin(diaper) dermatitis, Candida onychia and paronychia, candidosis of theexternal ear, and miscellaneous cutaneous forms of candidosis; chronicmucocutaneous candidosis; systemic candidosis; candidosis of thegastrointestinal tract, such as, esophageal candidosis, gastriccandidosis, candidosis of the intestine, Candida cholecystitis, and the"autobrewery syndrome" ("meitei-sho"); candidosis of the Urinary Tract,such as, renal candidosis (Candida pyelonephritis), candidosis of theurinary bladder (Candida cystitis), Candida urethritis and prostatitis;cardiovascular Candida infections, such as, candida endocarditis, andmyocarditis; Candida endocariditis, Candida myocarditis andpericarditis, Candida phlebitis and thrombophlebitis; Candida infectionsof the eye, such as, Candida endophthalmitis, Candida infections of thecornea and conjuctiva, and candidosis of the lacrimal sacs(dacryocystitis); candidosis of the central nervous system; Candidameningitis, cerebral candidosis, and candidosis of the inner ear;Candida infections of bones and joints; Candida peritonitis, hepatitisand miscellaneous other forms of systemic candidosis, such as, Candidaperitonitis, Candida hepatitis and splenitis, Candida pancreatitis,Congenital and intrauterine candidosis (Candida chorioamnionitis andfunisitis); disseminated candidosis, such as, in neonates, younginfants, and heroin addicts; Candida allergy, such as, cutaneous Candidaallergy and "candidids", Respiratory tract allergy to "Candida", Candidaallergy in other bodily sites, "chronic candidosis" and "the yeastconnection"; and also for assaying genetic variation; and foradministering a caYAE1 polypeptide or polynucleotide to an organism toraise an immunological response against a fungi, for example,opportunistic fungi, especially a Candida or Aspergillus.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to caYAE1 polynucleotide sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against caYAE1 polypeptides.

In accordance with another aspect of the present invention, there areprovided caYAE1 agonists which are also preferably bacteriostatic orbacteriocidal.

In accordance with yet another aspect of the present invention, thereare provided caYAE1 antagonists which are also preferably bacteriostaticor bacteriocidal.

In a further aspect of the invention there are provided compositionscomprising a caYAE1 polynucleotide or a caYAE1 polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the polynucleotide sequence of Candida albicans YAE1,caYAE1 SEQ ID NO:1!. The open reading frame of caYAE1 is from nucleotide201 to 737.

FIG. 2 shows the amino acid sequence of Candida albicans YAE1, caYAE1SEQ ID NO:2! deduced from the polynucleotide sequence of FIG. 1.

FIG. 3 shows the nucleotide sequence of the S. cerevisiae and C.albicans YAE1 genes which are 50% identical

FIG. 4 shows the amino acid sequence of the S. cerevisiae and C.albicans YAE1 proteins. The deduced protein sequence is shown in thisFigure. The caYae1p (from C. albicans) is 179 amino acids in length and42% identical, 60% similar to scYae1p (from S. ceriviseae) which is 121amino acids in length.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe Examples. The explanations are provided as a convenience and are notlimitative of the invention. caYAE1-BINDING MOLECULE, as used herein,refers to molecules or ions which bind or interact specifically withcaYAE1 polypeptides or polynucleotides of the present invention,including, for example enzyme substrates, cell membrane components andclassical receptors. Binding between polypeptides of the invention andsuch molecules, including binding or binding or interaction moleculesmay be exclusive to polypeptides of the invention, which is preferred,or it may be highly specific for polypeptides of the invention, which isalso preferred, or it may be highly specific to a group of proteins thatincludes polypeptides of the invention, which is preferred, or it may bespecific to several groups of proteins at least one of which includes apolypeptide of the invention. Binding molecules also include antibodiesand antibody-derived reagents that bind specifically to polypeptides ofthe invention.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a polynucleotide region that regulatesreplication, transcription or translation or other processes importantto expression of the polypeptide in a host cell, or a polynucleotidecomprising both a region that encodes a polypeptide and a regionoperably linked thereto that regulates expression. Genetic elements maybe comprised within a vector that replicates as an episomal element;that is, as a molecule physically independent of the host cell genome.They may be comprised within plasmids. Genetic elements also may becomprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

HOST CELL is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

IDENTITY or SIMILARITY, as known in the art, are relationships betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Both identityand similarity can be readily calculated (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity and similarity between two polynucleotideor two polypeptide sequences, both terms are well known to skilledartisans (Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonlyemployed to determine identity or similarity between sequences include,but are not limited to those disclosed in Carillo, H., and Lipman, D.,SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determineidentity are designed to give the largest match between the sequencestested. Methods to determine identity and similarity are codified incomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F.et al., J. Molec. Biol. 215: 403 (1990)).

ISOLATED means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living organismin its natural state is not "isolated," but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis "isolated", as the term is employed herein. As part of or followingisolation, such polynucleotides can be joined to other polynucleotides,such as DNAs, for mutagenesis, to form fusion proteins, and forpropagation or expression in a host, for instance. The isolatedpolynucleotides, alone or joined to other polynucleotides such asvectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions or single-, double- andtriple-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded, or a mixture of single- anddouble-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are "polynucleotides" as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. Polynucleotidesembraces short polynucleotides often referred to as oligonucleotide(s).

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature. Several particularly common modifications,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation, forinstance, are described in most basic texts, such as, for instancePROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York (1993). Many detailed reviews areavailable on this subject, such as, for example, those provided by Wold,F., Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N. Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, as is well known and as notedabove, that polypeptides are not always entirely linear. For instance,polypeptides may be generally as a result of posttranslational events,including natural processing event and events brought about by humanmanipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli or other cells, priorto proteolytic processing, almost invariably will be N-formylmethionine.During post-translational modification of the peptide, a methionineresidue at the NH₂ -terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing and themethionineless amino terminal variants of the protein of the invention.The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in fungal hosts such as, for example,S. ceriviseae. Accordingly, when glycosylation is desired, a polypeptideshould be expressed in a glycosylating host, generally a eukaryoticcell. Insect cell often carry out the same posttranslationalglycosylations as mammalian cells and, for this reason, insect cellexpression systems have been developed to express express efficientlymammalian proteins having native patterns of glycosylation, inter alia.Similar considerations apply to other modifications. It will beappreciated that the same type of modification may be present in thesame or varying degree at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications. In general,as used herein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesizedrecombinantly by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail. (1) A polynucleotide that differs in nucleotide sequence fromanother, reference polynucleotide. Generally, differences are limited sothat the nucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical. As noted below,changes in the nucleotide sequence of the variant may be silent. Thatis, they may not alter the amino acids encoded by the polynucleotide.Where alterations are limited to silent changes of this type a variantwill encode a polypeptide with the same amino acid sequence as thereference. Also as noted below, changes in the nucleotide sequence ofthe variant may alter the amino acid sequence of a polypeptide encodedby the reference polynucleotide. Such nucleotide changes may result inamino acid substitutions, additions, deletions, fusions and truncationsin the polypeptide encoded by the reference sequence, as discussedbelow. (2) A polypeptide that differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference and the variant are closely similaroverall and, in many region, identical. A variant and referencepolypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel caYAE1 polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel caYAE1 gene of Candida albicans, which isrelated by amino acid sequence homology to the YAE1 polypeptide ofSaccharomyces cerevisiae. The invention relates especially to caYAE1having the nucleotide and amino acid sequences set out in FIG. 1 andFIG. 2 respectively. The nucleotide and amino acid sequences set out inFIGS. 1 SEQ ID NO:1! and 2 SEQ ID NO:2! were obtained by sequencing theC. albicans DNA.

Techniques are available to evaluate temporal gene expression in fungi,particularly as it applies to viability under laboratory and hostinfection conditions. A number of methods can be used to identify geneswhich are essential to survival per se, or essential to theestablishment/maintenance of an infection. Identification of expressionof a sequence by one of these methods yields additional informationabout its function and permits the selection of such sequence forfurther development as a screening target. Briefly, these approachesinclude:

1) Differential display

This technique is described by Chuang et al., J. Bacteriol.175:2026-2036(1993), the contents of which is incorporated by referencefor background purposes. This method identifies those genes which areexpressed in an organism by identifying mRNA present usingrandomly-primed RT-PCR. By comparing pre-infection and post infectionprofiles, genes up and down regulated during infection can be identifiedand the RT-PCR product sequenced and matched to library sequences.

2) Generation of conditional lethal mutants by transposon mutagenesis.

This technique, described by de Lorenzo, V. et al., Gene 123:17-24(1993); Neuwald, A. F. et al., Gene 125: 69-73(1993); and Takiff, H. E.et al., J. Bacteriol. 174:1544-1553(1992), the contents of which isincorporated by reference for background purposes, identifies geneswhose expression are essential for cell viability.

In this technique transposons carrying controllable promoters, whichprovide transcription outward from the transposon in one or bothdirections, are generated. Random insertion of these transposons intotarget organisms and subsequent isolation of insertion mutants in thepresence of inducer of promoter activity ensures that insertions whichseparate promoter from coding region of a gene whose expression isessential for cell viability will be recovered. Subsequent replicaplating in the absence of inducer identifies such insertions, since theyfail to survive. Sequencing of the flanking regions of the transposonallows identification of site of insertion and identification of thegene disrupted. Close monitoring of the changes in cellularprocesses/morphology during growth in the absence of inducer yieldsinformation on likely function of the gene. Such monitoring couldinclude flow cytometry (cell division, lysis, redox potential, DNAreplication), incorporation of radiochemically labeled precursors intoDNA, RNA, protein, lipid, peptidoglycan, monitoring reporter enzyme genefusions which respond to known cellular stresses.

3) Generation of conditional lethal mutants by chemical mutagenesis.

This technique is described by Beckwith, J., Methods in Enzymology 204:3-18(1991), the contents of which are incorporated herein by referencefor background purposes. In this technique random chemical mutagenesisof target organism, growth at temperature other than physiologicaltemperature (permissive temperature) and subsequent replica plating andgrowth at different temperature (e.g., 42° C. to identify ts, 25° C. toidentify cs) are used to identify those isolates which now fail to grow(conditional mutants). As above close monitoring of the changes upongrowth at the non-permissive temperature yields information on thefunction of the mutated gene. Complementation of conditional lethalmutation by library from target organism and sequencing of complementinggene allows matching with library sequences.

4) RT-PCR

Fungal messenger RNA, preferably that of Candida albicans, is isolatedfrom fungal infected tissue e.g. 48 hour murine lung infections, and theamount of each MRNA species assessed by reverse transcription of the RNAsample primed with random hexanucleotides followed by PCR with genespecific primer pairs. The determination of the presence and amount of aparticular mRNA species by quantification of the resultant PCR productprovides information on the fungal genes which are transcribed in theinfected tissue. Analysis of gene transcription can be carried out atdifferent times of infection to gain a detailed knowledge of generegulation in fungal pathogenesis allowing for a clearer understandingof which gene products represent targets for screens for novelantifungals. Because of the gene specific nature of the PCR primersemployed it should be understood that the fungal mRNA preparation neednot be free of mammalian RNA. This allows the investigator to carry outa simple and quick RNA preparation from infected tissue to obtain fungalmRNA species which are very short lived in the bacterium (in the orderof 2 minute halflives). Optimally the fungal MRNA is prepared frominfected murine lung tissue by mechanical disruption in the presence ofTRIzole (GIBCO-BRL) for very short periods of time, subsequentprocessing according to the manufacturers of TRIzole reagent and DNAasetreatment to remove contaminating DNA. Preferably the process isoptimized by finding those conditions which give a maximum amount offungal 16S ribosomal RNA, preferably that of Candida albicans, asdetected by probing Northerns with a suitably labeled sequence specificoligonucleotide probe. Typically a 5' dye labelled primer is used ineach PCR primer pair in a PCR reaction which is terminated optimallybetween 8 and 25 cycles. The PCR products are separated on 6%polyacrylamide gels with detection and quantification using GeneScanner(manufactured by ABI).

5) Targeted gene disruption

This technique is described by Fonzi, W. A. and Irwin, M. Y. Genetics134: 717-728 (1993), the contents of which are incorporated herein byreference for background purposes. In this technique a selectable markerflanked by direct repeats is inserted into the gene of interest. Thisconstruct is then integrated into the genome by homologousrecombination, selecting for the expression of the marker and therebydisrupting the targeted gene. The marker gene can be removed byhomologous recombination of the flanking sequences leaving behind asingle copy of the direct repeat sequence which disrupts the targetedgene. Integration of the disruption cassette can then be repeatedallowing for disruption of multiple alleles of the targeted gene. Asabove close monitoring of the changes upon growth after gene disruptionyields information on the function of the mutated gene.

Each of these techniques may have advantages or disadvantage dependingon the particular application. The skilled artisan would choose theapproach that is the most relevant with the particular end use in mind.For example, some genes might be recognised as essential for infectionbut in reality are only necessary for the initiation of infection and sotheir products would represent relatively unattractive targets forantifungals developed to cure established and chronic infections.

Use of the of these technologies when applied to the sequences of thepresent invention enables identification of fungal proteins expressedduring infection, inhibitors of which would have utility in anti-fungaltherapy.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the caYAE1 polypeptidehaving the deduced amino acid sequence of FIG. 2 SEQ ID NO:2!.

This invention provides, among other things, that one of thehypothetical S. ceriviseae proteins disclosed in EMBO Journal 15,2031-2049, 1996, encoded by the ORF YJR067c, is essential for the growthof wild-type yeast. In view of this, strains were isolated which have anull chromosomal mutation in YJR067c, with viability being dependentupon the presence of a plasmid carrying a wild-type copy of YJR067c.This indicated that loss of the chromosomal YJR067c protein is lethal.This was confirmed by direct construction of a YJR067cdeletion/insertion mutation in a diploid strain, followed by sporulationand tetrad dissection; haploid YJR067c-deleted segregants were neverrecovered, indicating that YJR067c is an essential gene. The protein wastagged with a T7 epitope and a polypeptide of the expected size wasobserved by western analysis (YJR067c encodes a 141-amino acid protein).Reverse transcription PCR assays also confirmed that YJR067c is anexpressed gene (herein "scYAE1").

The scYAE1 coding region was found among a collection of randomly clonedand sequenced S. ceriviseae clones. Cross-species Southern blots ("zooblots") did not identify any potential mammalian homologues. A Candidaalbicans clone was, however, identified from a database of sequencefragments, which is a scYAE1 homologue sequence fragment (the homepageof the Candida albicans information pages on the Alces WWW server islocated at: http://alces.med.umn.edu/Candida.html available from themapping project at the University of Minnesota). The C. albicans clone(clone 2115) is 49% identical at the nucleotide level to scYAE1.Oligonucleotide probes were designed from the C. albicans sequence andused to isolate a full-length YAE1 homologue from a C. albicans genomiclibrary carried in the plasmid YEp13 (Mol. Gen. Genet. 200. 500-502,1985). Seven isolates representing 2 individual, overlapping clones wereisolated. Subdlones of these two clones were sequenced and shown tocontain an ORF encoding a 179 amino acid protein caYAE1 which is 42%identical, 60% similar to S. cerevisiae scYAE1 protein. The sequence ofthe C. albicans caYAE1 gene is shown in the attachment. The C. albicansclone is unable to complement the lethal phenotype of a scYAE1-deletionin S. cerevisiae, but this may be due to the inability of S. cerevisiaeto utilize the Candida promoter. Interestingly, complementation can beachieved using a plasmid carrying the S. cerevisiae scYAE1 coding regionunder control of the galactose-inducible GAL1 promoter even in thepresence of glucose, suggesting that very little scYAE1 is required tosupport viability.

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 SEQ ID NO:1!, a polynucleotide of the presentinvention encoding caYAE1 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning andsequencing chromosomal DNA fragments from Candida albicans B792 cells asstarting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide of the invention sequence, such asthat sequence given in FIG. 1 SEQ ID NO: 1! typically a library ofclones of chromosomal DNA of Candida albicans B792 in S. ceriviseae,Aspergillus, E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using high stringency washes. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see Screening By Hybridization1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70).Illustrative of the invention, the polynucleotide set out in FIG. 1 SEQID NO:1! was discovered in a DNA library derived from Candida albicansB792.

caYAE1 of the invention is structurally related to other proteins of theYAE1 family, as shown by the results of sequencing the DNA encodingcaYAE1. The DNA sequence thus obtained is set out in FIG. 1 SEQ IDNO:1!. It contains an open reading frame encoding a protein of havingabout the number of amino acid residues set forth in FIG. 2 SEQ ID NO:2!with a deduced molecular weight that can be calculated using amino acidresidue molecular weight values well known in the art. FIG. 3 shows asequence alignment. The nucleotide sequence of the ORFs of the S.cerevisiae and C. albicans caYAE1 genes are 50% identical. The portionof the caYAE1 sequence which was identified from a set of randomlysequenced Candida albicans clones (designated clone 2115) spans fromnucleotide 285 to 797. The original clone identified, 2115, lacked aportion of the 5' end of the open reading frame and had some gaps in thesequence.

The protein exhibits greatest homology to Saccharomyces cerevisiae YAE1protein among known proteins. caYAE1 of FIG. 2 SEQ ID NO:2! has about42% identity over its entire length and about 60% similarity over itsentire length with the amino acid sequence of scYAE1 polypeptide. FIG. 4shows an alignment of the caYAE1 and scYAE1 amino acid sequences.

Polynucleotides of the present invention may be in the form of RNA, suchas MRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide shown in FIG. 1 SEQ ID NO:1!.It also may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of FIG. 2 SEQ ID NO:2!.

Polynucleotides of the present invention which encode the polypeptide ofFIG. 2 SEQ ID NO:2! may include, but are not limited to the codingsequence for the mature polypeptide, by itself; the coding sequence forthe mature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to non-coding 5' and 3' sequences, such as the transcribed,non-translated sequences that play a role in transcription (includingtermination signals, for example), ribosome binding, mRNA stabilityelements, and additional coding sequence which encode additional aminoacids, such as those which provide additional functionalities. Thus, forinstance, the polypeptide may be fused to a marker sequence, such as apeptide, which facilitates purification of the fused polypeptide. Incertain embodiments of this aspect of the invention, the marker sequenceis a hexa-histidine peptide, such as the tag provided in the pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag may also be used tocreate fusion proteins and corresponds to an epitope derived ofinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37: 767 (1984), for instance. Polynucleotides of the inventionalso include, but are not limited to, polynucleotides comprising astructural gene and its naturally associated genetic elements.

In accordance with the foregoing, the term "polynucleotide encoding apolypeptide" as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlyfungal, and more particularly the Candida albicans YAE1 having the aminoacid sequence set out in FIG. 2 SEQ ID NO:2!. The term encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the polypeptide (for example, interrupted by insertionsequence, introns or editing) together with additional regions, thatalso may contain coding and/or non-coding sequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 2 SEQ ID NO:2!. A variant of the polynucleotide may be a naturallyoccurring variant such as a naturally occurring allelic variant, or itmay be a variant that is not known to occur naturally. Suchnon-naturally occurring variants of the polynucleotide may be made bymutagenesis techniques, including those applied to polynucleotides,cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of caYAE1 set out in FIG. 2 SEQ ID NO:2!; variants, analogs,derivatives and fragments thereof, and fragments of the variants,analogs and derivatives.

Further particularly preferred in this regard are polynucleotidesencoding caYAE1 variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of caYAE1 polypeptide of FIG. 2 SEQ ID NO:2! in whichseveral, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residuesare substituted, deleted or added, in any combination. Especiallypreferred among these are silent substitutions, additions and deletions,which do not alter the properties and activities of caYAE1. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are polynucleotides encoding polypeptides having theamino acid sequence of FIG. 2 SEQ ID NO:2!, without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding caYAE1 polypeptide having the amino acid sequence set out inFIG. 2 SEQ ID NO:2!, and polynucleotides which are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover their entire length to a polynucleotide encoding caYAE1 polypeptideof the Candida albicans DNA and polynucleotides complementary thereto.In this regard, polynucleotides at least 90% identical over their entirelength to the same are particularly preferred, and among theseparticularly preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Preferred embodiments in this respect, moreover, are polynucleotideswhich encode polypeptides which retain substantially the same biologicalfunction or activity as the mature polypeptide encoded by the DNA ofFIG. 1 SEQ ID NO:1!.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding caYAE1 andto isolate cDNA and genomic clones of other genes that have a highsequence similarity to the caYAE1 gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less. An exampleof a probe useful to detect the polynucleotide of the invention is5'-ggatttgatgatggatatcc-3' SEQ ID NO:3!.

For example, the coding region of the caYAE1 gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofcDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsof and diagnostics for disease, particularly human disease, as furtherdiscussed herein relating to polynucleotide assays, inter alia.

The polynucleotides of the invention that are oligonucleotides,including SEQ ID NOS:3 and 4, derived from the sequences of SEQ ID NO:1may be used in the processes herein as described, but preferably forPCR, to determine whether or not the Candida albicans genes identifiedherein in whole or in part are transcribed in infected tissue. It isrecognized that such sequences will also have utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may allowprotein transport, may lengthen or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Polypeptides

The present invention further relates to a caYAE1 polypeptide which hasa deduced amino acid sequence of 179 amino acids in length, as set forthin FIG. 2 SEQ ID NO:2!.

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms "fragment," "derivative" and "analog" whenreferring to the polypeptide of FIG. 2 SEQ ID NO:2!, means a polypeptidewhich retains essentially the same biological function or activity assuch polypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 2 SEQ IDNO:2! may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of caYAE1 set outin FIG. 2 SEQ ID NO:2!, variants, analogs, derivatives and fragmentsthereof, and variants, analogs and derivatives of the fragments.Alternatively, particularly preferred embodiments of the invention inthis regard are polypeptides having the amino acid sequence of thecaYAE1, variants, analogs, derivatives and fragments thereof, andvariants, analogs and derivatives of the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the caYAE1 polypeptide ofFIG. 2 SEQ ID NO:2!, in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the caYAE1. Also especially preferred in this regard areconservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequence of FIG. 2 SEQ ID NO:2! withoutsubstitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide ofFIG. 2 SEQ ID NO:2! (in particular the mature polypeptide) as well aspolypeptides which have at least 70% identity to the polypeptide of FIG.2 SEQ ID NO:2!, preferably at least 80% identity to the polypeptide ofFIG. 2 SEQ ID NO:2!, and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of FIG. 2 SEQ IDNO:2! and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the polypeptide of FIG. 2 SEQ IDNO:2! and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of caYAE1, most particularlyfragments of caYAE1 having the amino acid set out in FIG. 2 SEQ IDNO:2!, and fragments of variants and derivatives of the caYAE1 of FIG. 2SEQ ID NO:2!.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned caYAE1 polypeptides and variants or derivativesthereof.

Such fragments may be "free-standing," i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a caYAE1polypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the caYAE1fragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from caYAE1.

Representative examples of polypeptide fragments of the invention,include, for example, fragments from amino acid number 1-20, 21-40,41-60, 61-80, 81-100, and 101-120, 121-140, 141-160, 161-179, and anycombination of these amino acid fragments.

In this context "about" herein includes the particularly recited rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes.

Preferred fragments of the invention include, for example, truncationpolypeptides of caYAE1. Truncation polypeptides include caYAE1polypeptides having the amino acid sequence of FIG. 2, or of variants orderivatives thereof, except for deletion of a continuous series ofresidues (that is, a continuous region, part or portion) that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus or, as in double truncation mutants, deletion of twocontinuous series of residues, one including the amino terminus and oneincluding the carboxyl terminus. Fragments having the size ranges setout about also are preferred embodiments of truncation fragments, whichare especially preferred among fragments generally. Degradation forms ofthe polypeptides of the invention in a host cell, particularly aCandida, are also preferred.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of caYAE1.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and tum-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions of caYAE1, and combinations of such fragments.

Preferred regions are those that mediate activities of caYAE1. Mosthighly preferred in this regard are fragments that have a chemical,biological or other activity of caYAE1, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Further preferred polypeptide fragments are those that areantigenic or immunogenic in an animal, especially in a human.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespond to the preferred fragments, as discussed above.

Vectors, host cells, expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, host cellswhich are genetically engineered with vectors of the invention and theproduction of polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. Introduction of apolynucleotides into the host cell can be affected by calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Polynucelotide constructs in host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Plasmids generallyare designated herein by a lower case p preceded and/or followed bycapital letters and/or numbers, in accordance with standard namingconventions that are familiar to those of skill in the art. Startingplasmids disclosed herein are either commercially available, publiclyavailable, or can be constructed from available plasmids by routineapplication of well known, published procedures. Many plasmids and othercloning and expression vectors that can be used in accordance with thepresent invention are well known and readily available to those of skillin the art.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom fungal plasmids, from bacteriophage, from transposons, from yeastepisomes, from insertion elements, from yeast chromosomal elements, fromviruses such as baculoviruses, papova viruses, such as SV40, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such asthose derived from plasmid and bacteriophage genetic elements, such ascosmids and phagemids, all may be used for expression in accordance withthis aspect of the present invention. Generally, any vector suitable tomaintain, propagate or express polynucleotides to express a polypeptidein a host may be used for expression in this regard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques, such as, for example,those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude, but are not limited to, the phage lambda PL promoter, the E.coli lac, trp and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating codon, for example, AUG, at the beginning and a terminationcodon appropriately positioned at the end of the polypeptide to betranslated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as transcription factors, repressor binding sitesand termination, among others.

Vectors for propagation and expression generally will include selectablemarkers and amplification regions, such as, for example, those set forthin Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia, and pBR322 (ATCC 37017). Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG availablefrom Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. These vectors are listed solely by way of illustration of themany commercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase ("CAT") transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable, such as pKK232-8 and pCM7. Promoters for expression ofpolynucleotides of the present invention include not only well known andreadily available promoters, but also promoters that readily may beobtained by the foregoing technique, using a reporter gene.

Among known prokaryotic promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacd and lacZ and promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are the CMVimmediate early promoter, the HSV thymidine kinase promoter, the earlyand late SV40 promoters, the promoters of retroviral LTRs, such as thoseof the Rous sarcoma virus ("RSV"), and met allothionein promoters, suchas the mouse met allothionein-I promoter.

Recombinant expression vectors will include, for example, origins ofreplication, a promoter preferably derived from a highly-expressed geneto direct transcription of a downstream structural sequence, and aselectable marker to permit isolation of vector containing cells afterexposure to the vector.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5' to aribosome binding site. The ribosome binding site will be 5' to the codonthat initiates translation of the polypeptide to be expressed, forexample AUG or GUG. Generally, there will be no other open readingframes that begin with an initiation codon, usually AUG, and lie betweenthe ribosome binding site and the initiation codon. Also, generally,there will be a translation stop codon at the end of the polypeptide andthere will be a polyadenylation signal in constructs for use ineukaryotic hosts. Transcription termination signal appropriatelydisposed at the 3' end of the transcribed region may also be included inthe polynucleotide construct.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N- or C-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunolglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobin molecules together with another proteinor part thereof. In drug discovery, for example, proteins have beenfused with antibody Fc portions for the purpose of high-throughputscreening assays to identify antagonists. See, D. Bennett et al.,Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson etal., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471(1995).

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Mammalian expression vectors may comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation regions, splice donor and acceptor sites,transcriptional termination sequences, and 5' flanking non-transcribedsequences that are necessary for expression. caYAE1 polypeptide can berecovered and purified from recombinant cell cultures by well-knownmethods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingprotein may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Polynucleotide assays

This invention is also related to the use of the caYAE1 polynucleotidesto detect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of caYAE1 in a eukaryote, particularly amammal, and especially a human, will provide a diagnostic method fordiagnosis of a disease. Eukaryotes (herein also "individual(s)"),particularly mammals, and especially humans, infected with an organismcomprising the caYAE1 gene may be detected at the DNA level by a varietyof techniques. Nucleic acids for diagnosis may be obtained from aninfected individual's cells and tissues, such as bone, blood, muscle,cartilage, and skin. Genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986) prior to analysis. RNA or cDNA may also be used in thesame ways. As an example, PCR primers complementary to the nucleic acidencoding caYAE1 can be used to identify and analyze caYAE1 presenceand/or expression. Using PCR, characterization of the strain ofprokaryote present in a eukaryote, particularly a mammal, and especiallya human, may be made by an analysis of the genotype of the prokaryotegene. For example, deletions and insertions can be detected by a changein size of the amplified product in comparison to the genotype of areference sequence. Point mutations can be identified by hybridizingamplified DNA to radiolabeled caYAE1 RNA or alternatively, radiolabeledcaYAE1 antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic characterization based on DNA sequence differences may beachieved by detection of alteration in electrophoretic mobility of DNAfragments in gels, with or without denaturing agents. Small sequencedeletions and insertions can be visualized by high resolution gelelectrophoresis. DNA fragments of different sequences may bedistinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science, 230: 1242(1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms (RFLP) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Cells carrying mutations or polymorphisms in the gene of the presentinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred o usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to the nucleicacid encoding caYAE1 can be used to identify and analyze mutations.Examples of representative primers are shown below in Table 1.

                  TABLE 1    ______________________________________    Primers used for amplification of caYAE1 polynucleotides    SEQ ID NO        PRIMER SEQUENCE    ______________________________________    3                5'-ggatttgatgatggatatcc-3'    4                5'-aattcctttttcgcttcattaaat-3'    ______________________________________

These primers may be used for amplifying caYAE1 DNA isolated from asample derived from an individual. The invention also provides theprimers of Table 1 with 1, 2, 3 or 4 nucleotides removed from the 5'and/or the 3' end. The primers may be used to amplify the gene isolatedfrom the individual such that the gene may then be subject to varioustechniques for elucidation of the DNA sequence. In this way, mutationsin the DNA sequence may be diagnosed.

The invention provides a process for diagnosing, disease, preferablyfungal infections, more preferably Candida albicans infections, and mostpreferably candidosis (candidiasis), for example, superficialcandidosis; candidosis of the oropharynx, such as, oral thrush (acutepseudomembraneous candodisis), denture stomatitis, angular choilitis(perleche), Candida leukoplakia (chronic hyperplastic candidosis),midline glossitis (median rbomboid glossitis, glossal central papillaryatrophy, "antibiotic sore tongue", acute atrophic candidosis), andmiscellaneous forms of oral candidosis; candidosis of the genitalia,such as, vulvovaginal candidosis (vaginal thrush, Candida colpitis), andcandidosis of the penis (Candida balanitis, balanoposthitis andurethritis); candidosis of the skin, nails and other external sites,such as, Candida intertrigo, napkin (diaper) dermatitis, Candida onychiaand paronychia, candidosis of the external ear, and miscellaneouscutaneous forms of candidosis; chronic mucocutaneous candidosis;systemic candidosis; candidosis of the gastrointestinal tract, such as,esophageal candidosis, gastric candidosis, candidosis of the intestine,Candida cholecystitis, and the "autobrewery syndrome" ("meitei-sho");candidosis of the Urinary Tract, such as, renal candidosis (Candidapyelonephritis), candidosis of the urinary bladder (Candida cystitis),Candida urethritis and prostatitis; cardiovascular Candida infections,such as, candida endocarditis, and myocarditis; Candida endocariditis,Candida myocarditis and pericarditis, Candida phlebitis andthrombophlebitis; Candida infections of the eye, such as, Candidaendophthalmitis, Candida infections of the cornea and conjuctiva, andcandidosis of the lacrimal sacs (dacryocystitis); candidosis of thecentral nervous system; Candida meningitis, cerebral candidosis, andcandidosis of the inner ear; Candida infections of bones and joints;Candida peritonitis, hepatitis and miscellaneous other forms of systemiccandidosis, such as, Candida peritonitis, Candida hepatitis andsplenitis, Candida pancreatitis, Congenital and intrauterine candidosis(Candida chorioamnionitis and funisitis); disseminated candidosis, suchas, in neonates, young infants, and heroin addicts; Candida allergy,such as, cutaneous Candida allergy and "candidids", Respiratory tractallergy to "Candida", Candida allergy in other bodily sites, "chroniccandidosis" and "the yeast connection," comprising determining from asample derived from an individual an increased level of expression ofpolynucleotide having the sequence of FIG. 1 SEQ ID NO: 1!. Increasedexpression of caYAE1 polynucleotide can be measured using any one of themethods well known in the art for the quantitation of polynucleotides,such as, for example, PCR, RT-PCR, RNase protection, Northern blottingand other hybridization methods.

Polypeptide assays

The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of caYAE1protein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of caYAE1 proteincompared to normal control tissue samples may be used to detect thepresence of an infection, for example. Assay techniques that can be usedto determine levels of a caYAE1 protein, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays. Among these ELISAs frequently are preferred. An ELISAassay initially comprises preparing an antibody specific to caYAE1,preferably a monoclonal antibody. In addition a reporter antibodygenerally is prepared which binds to the monoclonal antibody. Thereporter antibody is attached a detectable reagent such as radioactive,fluorescent or enzymatic reagent, in this example horseradish peroxidaseenzyme.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. The present invention includes, for examplesmonoclonal and polyclonal antibodies, chimeric, single chain, andhumanized antibodies, as well as Fab fragments, or the product of an Fabexpression library.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique known in the artwhich provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

Alternatively phage display technology could be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty, J.et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, antibodies against caYAE1 may be employed to inhibitand/or treat infections, particularly fungal infections and especiallycandidosis (candidiasis), for example, superficial candidosis;candidosis of the oropharynx, such as, oral thrush (acutepseudomembraneous candodisis), denture stomatitis, angular choilitis(perleche), Candida leukoplakia (chronic hyperplastic candidosis),midline glossitis (median rbomboid glossitis, glossal central papillaryatrophy, "antibiotic sore tongue", acute atrophic candidosis), andmiscellaneous forms of oral candidosis; candidosis of the genitalia,such as, vulvovaginal candidosis (vaginal thrush, Candida colpitis), andcandidosis of the penis (Candida balanitis, balanoposthitis andurethritis); candidosis of the skin, nails and other external sites,such as, Candida intertrigo, napkin (diaper) dermatitis, Candida onychiaand paronychia, candidosis of the external ear, and miscellaneouscutaneous forms of candidosis; chronic mucocutaneous candidosis;systemic candidosis; candidosis of the gastrointestinal tract, such as,esophageal candidosis, gastric candidosis, candidosis of the intestine,Candida cholecystitis, and the "autobrewery syndrome" ("meitei-sho");candidosis of the Urinary Tract, such as, renal candidosis (Candidapyelonephritis), candidosis of the urinary bladder (Candida cystitis),Candida urethritis and prostatitis; cardiovascular Candida infections,such as, candida endocarditis, and myocarditis; Candida endocariditis,Candida myocarditis and pericarditis, Candida phlebitis andthrombophlebitis; Candida infections of the eye, such as, Candidaendophthalmitis, Candida infections of the cornea and conjuctiva, andcandidosis of the lacrimal sacs (dacryocystitis); candidosis of thecentral nervous system; Candida meningitis, cerebral candidosis, andcandidosis of the inner ear; Candida infections of bones and joints;Candida peritonitis, hepatitis and miscellaneous other forms of systemiccandidosis, such as, Candida peritonitis, Candida hepatitis andsplenitis, Candida pancreatitis, Congenital and intrauterine candidosis(Candida chorioamnionitis and funisitis); disseminated candidosis, suchas, in neonates, young infants, and heroin addict; Candida alergy, suchas cutaneous Candida allergy and "candidids", Respiratory tract allergyto "Candida", Candida alergy in other bodily sites, "chronic candidosis"and "the yeast connection."

Polypeptide derivatives include antigenically, epitopically orimmunologically equivalent derivatives which form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognised by certain antibodies which, when raised to theprotein or polypeptide according to the present invention, interferewith the immediate physical interaction between pathogen and mammalianhost. The term "immunologically equivalent derivative" as used hereinencompasses a peptide or its equivalent which when used in a suitableformulation to raise antibodies in a vertebrate, the antibodies act tointerfere with the immediate physical interaction between pathogen andmammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation , with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody , for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem1989:264,16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS, 1986:83,9551), encapsulation of DNA invarious forms of liposomes (Kaneda et al., Science 1989:243,375),particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun etal., DNA Cell Biol 1993, 12:791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS 1984:81,5849).

caYAE1-binding molecules and assays

This invention also provides a method for identification of molecules,such as binding molecules, that bind caYAE1. Genes encoding proteinsthat bind caYAE1, can be identified by numerous methods known to thoseof skill in the art, for example, ligand panning and FACS sorting. Suchmethods are described in many laboratory manuals such as, for instance,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).Also, a labeled ligand can be photoaffinity linked to a cell extract.Polypeptides of the invention also can be used to assess caYAE1 bindingcapacity of caYAE1-binding molecules, in cells or in cell-freepreparations.

Polypeptides of the invention may also be used to assess the binding orsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics.

Antagonists and agonists--assays and molecules The invention alsoprovides a method of screening compounds to identify those which enhance(agonist) or block (antagonist) the action of caYAE1 polypeptides orpolynucleotides, such as its interaction with caYAE1-binding molecules.

For example, to screen for agonists or antagonists, a synthetic reactionmix, a cellular compartment, such as a membrane, cell envelope or cellwall, or a preparation of any thereof, may be prepared from a cell thatexpresses a molecule that binds caYAE1. The preparation is incubatedwith labeled caYAE1 in the absence or the presence of a candidatemolecule which may be a caYAE1 agonist or antagonist. The ability of thecandidate molecule to bind the binding molecule is reflected indecreased binding of the labeled ligand. Molecules which bindgratuitously, i.e., without inducing the effects of caYAE1 on bindingthe caYAE1 binding molecule, are most likely to be good antagonists.Molecules that bind well and elicit effects that are the same as orclosely related to caYAE1 are agonists. caYAE1-like effects of potentialagonists and antagonists may by measured, for instance, by determiningactivity of a reporter system following interaction of the candidatemolecule with a cell or appropriate cell preparation, and comparing theeffect with that of caYAE1 or molecules that elicit the same effects ascaYAE1. Reporter systems that may be useful in this regard include butare not limited to colorimetric labeled substrate converted intoproduct, a reporter gene that is responsive to changes in caYAE1activity, and binding assays known in the art.

Another example of an assay for caYAE1 antagonists is a competitiveassay that combines caYAE1 and a potential antagonist withmembrane-bound caYAE1-binding molecules, recombinant caYAE1 bindingmolecules, natural substrates or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. caYAE1 can be labeled, such as by radioactivity or a colorimetriccompound, such that the number of caYAE1 molecules bound to a bindingmolecule or converted to product can be determined accurately to assessthe effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducingcaYAE1-induced activities, thereby preventing the action of caYAE1 byexcluding caYAE1 from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules).

Preferred potential antagonists include compounds related to andderivatives of caYAE1.

In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the initial physical interaction between a pathogen and mammalianhost responsible for sequelae of infection. In particular the moleculesof the invention may be used: i) in the prevention of adhesion of fungi,in particular gram positive fungi, to mammalian extracellular matrixproteins on in-dwelling devices or to extracellular matrix proteins inwounds; ii) to block essential protein mediated mammalian cell invasionby, for example, initiating phosphorylation of mammalian tyrosinekinases (Rosenshine et al., Infect. Immun. 60:2211 (1992); iii) to blockfungal adhesion between mammalian extracellular matrix proteins andfungal essential proteins which mediate tissue damage; iv) to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

Each of the DNA sequences provided herein may be used in the discoveryand development of antifungal compounds. The encoded protein uponexpression can be used as a target for the screening of antifungaldrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective MRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The antagonists and agonists may be employed for instance to inhibitcandidosis (candidiasis), for example, superficial candidosis;candidosis of the oropharynx, such as, oral thrush (acutepseudomembraneous candodisis), denture stomatitis, angular choilitis(perleche), Candida leukoplakia (chronic hyperplastic candidosis),midline glossitis (median rbomboid glossitis, glossal central papillaryatrophy, "antibiotic sore tongue", acute atrophic candidosis), andmiscellaneous forms of oral candidosis; candidosis of the genitalia,such as, vulvovaginal candidosis (vaginal thrush, Candida colpitis), andcandidosis of the penis (Candida balanitis, balanoposthitis andurethritis); candidosis of the skin, nails and other external sites,such as, Candida intertrigo, napkin (diaper) dermatitis, Candida onychiaand paronychia, candidosis of the external ear, and miscellaneouscutaneous forms of candidosis; chronic mucocutaneous candidosis;systemic candidosis; candidosis of the gastrointestinal tract, such as,esophageal candidosis, gastric candidosis, candidosis of the intestine,Candida cholecystitis, and the "autobrewery syndrome" ("meitei-sho");candidosis of the Urinary Tract, such as, renal candidosis (Candidapyelonephritis), candidosis of the urinary bladder (Candida cystitis),Candida urethritis and prostatitis; cardiovascular Candida infections,such as, candida endocarditis, and myocarditis; Candida endocariditis,Candida myocarditis and pericarditis, Candida phlebitis andthrombophlebitis; Candida infections of the eye, such as, Candidaendophthalmitis, Candida infections of the cornea and conjuctiva, andcandidosis of the lacrimal sacs (dacryocystitis); candidosis of thecentral nervous system; Candida meningitis, cerebral candidosis, andcandidosis of the inner ear; Candida infections of bones and joints;Candida peritonitis, hepatitis and miscellaneous other forms of systemiccandidosis, such as, Candida peritonitis, Candida hepatitis andsplenitis, Candida pancreatitis, Congenital and intrauterine candidosis(Candida chorioamnionitis and funisitis); disseminated candidosis, suchas, in neonates, young infants, and heroin addicts; Candida allergy,such as, cutaneous Candida allergy and "candidids", Respiratory tractallergy to "Candida", Candida allergy in other bodily sites, "chroniccandidosis" and "the yeast connection."

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with caYAE1, or a fragment orvariant thereof, adequate to produce antibody to protect said individualfrom infection, particularly fungal infection and most particularlyCandida infections. Yet another aspect of the invention relates to amethod of inducing immunological response in an individual whichcomprises, through gene therapy, delivering gene encoding caYAE1, or afragment or a variant thereof, for expressing caYAE1, or a fragment or avariant thereof in vivo in order to induce an immunological response toproduce antibody to protect said individual from disease.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable or having inducedwithin it an immunological response, induces an immunological responsein such host to a caYAE1 or protein coded therefrom, wherein thecomposition comprises a recombinant caYAE1 or protein coded therefromcomprising DNA which codes for and expresses an antigen of said caYAE1or protein coded therefrom.

The caYAE1 or a fragment thereof may be fused with co-protein which maynot by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. Thus fused recombinant protein, preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of fungal cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Candida albicans will be particularly usefulfor identifying protein epitopes able to provoke a prophylactic ortherapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of Candida albicans infection inmammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of fungi, forexample by blocking adherence of fungi to damaged tissue. Examples oftissue damage include wounds in skin or connective tissue caused e.g. bymechanical, chemical or thermal damage or by implantation of indwellingdevices, or wounds in the mucous membranes, such as the mouth, mammaryglands, urethra or vagina.

The present invention also includes a vaccine formulation whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain caYAE1,it is to be understood that this covers fragments of the naturallyoccurring protein and similar proteins with additions, deletions orsubstitutions which do not substantially affect the immunogenicproperties of the recombinant protein.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about Img/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters,etc.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant fungi shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent Candida wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antifungal prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antifungals in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of fungi to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antifungal prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response.

A suitable unit dose for vaccination is 0.5-5 μg/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks.

With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable individuals.

The antibodies described above may also be used as diagnostic reagentsto detect the presence of fungi containing the essential protein.

In order to facilitate understanding of the following example certainfrequently occurring methods and/or terms will be described.

EXAMPLES

The present invention is further described by the following examples.These exemplification's, while illustrating certain specific aspects ofthe invention, do not portray the limitations or circumscribe the scopeof the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Example 1

Library Production

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Candida albicansstrain B792 in YEp13. In some cases the sequencing data from two or moreclones containing overlapping Candida albicans DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example: Methods 1, 2 and 3 below.

Total cellular DNA is isolated from Candida albicans B792 according tostandard procedures and size-fractionated by either of two methods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl2351), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Method 3

Total cellular DNA is partially hydrolyzed with Sau3A to generatefragments for cloning into library vectors, and such fragments aresize-fractionated according to standard procedures. These fragments arethen ligated into the BamHI site of the vector, such as YEp13. Theligated mixture is used to transform E. coli and then amplified bystandard procedures.

Example 2

scYAE1 Characterization

The scYAE1 gene was disrupted in a diploid strain and replaced by aselectable marker, HIS3, using homologous recombination. Theheterozygous diploid was sporulated and viability was shown to go 2:2.Those spores which did survive were all negative for the selectablemarker indicating that the disruption of scYAE1 resulted in inviability.The diploid was then transformed with a plasmid carrying scYAE1 and theselectable marker URA3 or ADE2. Spores were then rescued which had beendeleted for the genomic copy of scYAE1 and which carried the scYAE1 geneon a plasmid. The strains carrying scYAE1 on a plasmid marked with URA3were unable to lose the plasmid as shown by an inability to grow on5-FOA. Only strains which are URA3 minus are able to grow in thepresence of 5-FOA. The strains carrying scYAE1 on an ADE2 marked plasmidwere also not able to lose the plasmid as shown by non-sectoring whiteplasmids. If the plasmid were lost cells would show the ade2 minusphenotype of accumulation of a red pigment (Methods in Enzymology 194,281-318, 1991).

S. cerevisiae YAE1 protein is tagged and this tagged version is used inimmunofluorescence studies to determine the cellular localization of theprotein. Conditional alleles of scYAE1 may be identified and onceidentified are used as a basis for cell based sceens for anti-fungalcompounds.

Example 3

caYAE1 Characterization

The caYAE1 gene on a YEp13 plasmid which replicates in S. cerevisiae andwhich carries the selectable marker LEU2, was introduced into S.Cerevisiae strains in which the genomic scYAE1 was deleted and growthwas dependent upon a plasmid carrying scYAE1. These transformants werethen assayed for the ability to lose the plasmid carrying the scYAE1gene either by growth on 5-FOA or by accumulation of a red pigment. Ifthe strain is able to grow in the absence of the plasmid carrying scYAE1this would show that the caYAE1 gene was able to complement the scYAE1deletion. No complementation was observed by these assays but this isbelieved to be due to the inability of S. cerevisiae to utilize the C.albicans promoter. The caYAE1 gene was therefore expressed from the S.cerevisiae CUP1 promoter (Gene 48, 13-22,1986) and complementationassays were performed as described above. When expressed from a S.cerevisiae promoter, such as that of CUP1, the caYAE1 gene is able tocomplement deletion of the scYAE1 gene.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 797 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Genomic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE:    (vi) ORIGINAL SOURCE:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GTGCACAAGTTATCAATTTGTTTATTATTATAATTACACGGTAAAATCCCAGTTTTCTTC60    TTAAACAATCTTCTTCTTCTTGTTCTTCTAACATACAATTTTATTTCAATTCTACTTATA120    ACTTGACTCATTTGATTCCAATATTATACTAATACAGTCTCTATACTTTCTTTTGCTTTT180    CTTTTTTTTTTTTTTTCATAATGACATGTCAAGAGGATTGTTCATGTAAGAATAATGAAG240    CCCCCACAACAAAGACAACTGCCACCACAACTAATGTTGGTGATGGCCCTGGCCCTGGCC300    CTATCCCTGGCAATAATGATGATGTTGATGATGACATTTGGTCAGATGATGATACGAAAC360    TAATACCTCAAAATGATATAATACGATCACATTATAAAAAAGGGTATGTTGATGGGATAA420    CTCAAGCTAAAGAATCTTCATTACAACAAGGATTTGATGATGGATATCCTGAAGGTGCAA480    AATTAGGGATTAAAGTTGGTGAAATTTTAGCAAATTTAATCAATCAATGTAAAGATAGAA540    ACAGACAAGGAGATGATGATGATGATGATGATGAACAACTGGTAAGATTTAATGAAGCGA600    AAAAGGAATTGAATATAGTTAATGTTTTAAAAAAATCTTATTTTGATGAAGATTTGAATT660    TGAAAAAGAGTAATGGCAATAAAGAAACTGATGAATCATATCATGAATTAATTAATAAAT720    GGGAGAATGAAATGAAATGAAATGACAGTAAATTAAAATAGTATGTATAGATATAGAAGT780    TGAAGGAATTACTACTA797    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 179 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vi) ORIGINAL SOURCE:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetThrCysGlnGluAspCysSerCysLysAsnAsnGluAlaProThr    151015    ThrLysThrThrAlaThrThrThrAsnValGlyAspGlyProGlyPro    202530    GlyProIleProGlyAsnAsnAspAspValAspAspAspIleTrpSer    354045    AspAspAspThrLysLeuIleProGlnAsnAspIleIleArgSerHis    505560    TyrLysLysGlyTyrValAspGlyIleThrGlnAlaLysGluSerSer    65707580    LeuGlnGlnGlyPheAspAspGlyTyrProGluGlyAlaLysLeuGly    859095    IleLysValGlyGluIleLeuAlaAsnLeuIleAsnGlnCysLysAsp    100105110    ArgAsnArgGlnGlyAspAspAspAspAspAspAspGluGlnLeuVal    115120125    ArgPheAsnGluAlaLysLysGluLeuAsnIleValAsnValLeuLys    130135140    LysSerTyrPheAspGluAspLeuAsnLeuLysLysSerAsnGlyAsn    145150155160    LysGluThrAspGluSerTyrHisGluLeuIleAsnLysTrpGluAsn    165170175    GluMetLys    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Genomic DNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE:    (vi) ORIGINAL SOURCE:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GGATTTGATGATGGATATCC20    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE:    (vi) ORIGINAL SOURCE:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    AATTCCTTTTTCGCTTCATTAAAT24    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide encoding a polypeptidecomprising the amino acids of SEQ ID NO:2.
 2. A vector comprising thenucleotide of claim
 1. 3. A host cell comprising the vector of claim 2.4. A process for producing a polypeptide comprising the step ofexpressing from the host cell of claim 3 a polypeptide encoded by saidDNA.
 5. A process for producing a cell which expresses a polypeptidecomprising the step of transforming or transfecting the cell with thevector of claim 2 such that the cell expresses the polypeptide encodedby the nucleotides contained in the vector.
 6. The isolatedpolynucleotide of SEQ ID NO:1.
 7. An isolated polynucleotide comprisingnucleotides 201 through 737 inclusive as set forth in SEQ ID NO:1.
 8. Anisolated polynucleotide consisting of nucleotides 201 through 737inclusive as set forth in SEQ ID NO:1.
 9. An isolated polynucleotidecomprising nucleotides 1 through 797 inclusive as set forth in SEQ IDNO:1.
 10. An isolated polynucleotide consisting of nucleotides 1 through797 inclusive as set forth in SEQ ID NO:1.
 11. A probe comprising anisolated polynucleotide selected from the group consisting of: at least30 bases of the polynucleotide sequence set forth in SEQ ID NO: 1 and atleast 50 bases of the polynucleotide sequence set forth in SEQ ID NO: 1.12. A probe consisting of an isolated polynucleotide selected from thegroup consisting of: at least 30 bases of the polynucleotide sequenceset forth in SEQ ID NO: 1 and at least 50 bases of the polynucleotidesequence set forth in SEQ ID NO:
 1. 13. A vector comprising thepolynucleotide of claims 7 or
 9. 14. A host cell comprising the vectorof claim
 13. 15. A process for producing a response regulatorpolypeptide comprising the step of culturing a host cell of claim 14under conditions sufficient for the production of said polypeptide. 16.A polynucleotide which is fully complementary to a polynucleotide ofclaims 1, 3, 4, 5, 6, 11 and 12.